This invention relates to the synthesis of heterologous polypeptides in recombinant host cells. In particular, this relates to the secretion of mature human plasminogen activator proteases from bacterial transformants.
Inducible promoters are well known. Such promoters are characterized by the ability to increase the rate of transcription of genes under their control upon a change in the environment of the cell, typically the concentration of inorganic ions or nutrients, or a change in a physical condition such as the temperature of cultivation. Such promoters include the E. coli trp, E. coli lac and yeast acid phosphate, respectively, or the promoter controlled by the temperature sensitive lambda phage repressor (Harris, et al., infra). Inducible promoters have been employed to control the transcription of heterologous genes in recombinant cell culture. In this context, they serve to restrain transcription, and hence translation, of heterologous proteins that are known to be toxic to host cell or which are degraded proteolytically by the host cell. Induction from the promoter at a suitable phase in the recombinant culture therefore minimizes the contact of the host cell with the heterologous polypeptide, and vice versa.
Currently available inducible promoters all exhibit various constitutive levels of transcription, even when conditions are optimized for repression or deactivation of the promoter. For most recombinant systems this is not disadvantageous at the constitutive levels encountered because polypeptide losses to degradation are tolerable, toxicity is insufficient to seriously affect yields of heterologous polypeptide and/or polypeptide shunted into refractile bodies or other processing sinks is readily reactivated or reprocessed.
In an attempt to provide more control over induction of transcription of heterologous genes the T7 RNA polymerase system has been adapted to the synthesis of certain heterologous polypeptides in recombinant host cells.
This system, which has heretofore been extensively described per se. consists of an RNA polymerase that recognizes unique promoters present only in T7 DNA. None of these promoters are known to be transcribed by wild-type E. coli or mammalian RNA polymerases. This transcriptional system containing T7 RNA polymerase and the companion promoters is therefore highly selective, and the selectivity is enhanced by placing the T7 RNA polymerase gene under the control of the lac UV5 inducible promoter recognized by the host cell RNA polymerase (Studier et al., "J. Mol. Biol." 189:113-130 [1986]; Tabor et al., "Proc. Natl. Acad. Sci. USA" 82:1074-1078 [1985]). Tabor et al., (op. cit.), finding that expression of T7 RNA polymerase was excessive in their system, inserted a transcriptional terminator 5' to the polymerase gene in order to suppress its constitutive expression. This also reduced the inductive capability of the system. A mammalian cell expression system for recombinant cytoplasmic proteins using the T7 RNA polymerase has been described by Fuerst et al., "Proc. Natl. Acad. Sci. USA" 83:8122-8126 (1986). This system as presently constituted, notwithstanding its promise for improving the control of transcription from genes under its aegis, has not proven useful for controlling the expression of certain polypeptides, nor have conventional inducible promoters. In each instance, the constitutive level of transcription from these conventional systems has proven to be incompatible with the host cell and stability of the gene encoding the heterologous protein. The reasons for this incompatibility have been poorly understood. Typically, the art has simply observed that secretion of the desired protein was poor or nonexistent without delving into the mechanisms responsible.
Many heterologous polypeptides have been secreted from microbial hosts. Typically, a chimeric gene is constructed in which a signal sequence is ligated 5' to the codon for the mature amino terminus of the desired polypeptide. This gene is used to transform microbial host, the host cultured, and the secreted mature polypeptide recovered from the host cell periplasm or, occasionally, the culture medium. Representative general methods for the secretion of heterologous polypeptides from bacteria and yeast are described in Gilbert et al., U.S. Pat. No. 4,338,397 and EP 127,304 or EP 88,632, respectively. It is known to use signal sequences obtained from the host cell ("host-homologous signals"), or signals that are heterologous to the host cell (including the use of variant signals or signals which are native to the heterologous polypeptide). More recently, signals have been directly linked to the mature N-terminus of the heterologous polypeptide, although it also is conventional to link the heterologous polypeptide, together with a portion of its own signal, to the N-terminus of a host-homologous signal. In the alternative, heterologous polypeptide has been linked to the C-terminus of a host-homologous signal containing a portion of the homologous protein normally secreted under the control of the homologous signal.
Previous attempts to provide a microbial host-vector system for the secretion of human plasminogen activator have been unsuccessful, notwithstanding that commercial quantities of tissue type human plasminogen activator (t-PA) are readily secreted from transformants of mammalian cell lines. Bacteria, which do not glycosylate proteins, were first used as hosts for the recombinant synthesis of t-PA. See EP 93,619 and Harris et al. ("Mol. Biol. Med." 3:279-292 [1986]). Harris et al. describe the expression in E. coli of t-PA from a gene encoding N-methionyl t-PA. High levels of recombinant met t-PA did in fact accumulate in the Harris et al. E. coli hosts as insoluble refractile bodies, but little in the way of active enzyme could be recovered, despite extensive efforts to reactivate the enzyme by in vitro processing of the refractile bodies. These authors suggested that expression systems using vectors designed for secretion of proteins in E. coli or yeast may be more successful for making active enzyme under conditions where inclusion bodies are not formed. To date, this prophecy has proven equivocal in the case of yeast and false in the case of bacteria.
Lemontt et al. ("DNA" 4(6):419 [1985]) transformed yeast with t-PA preproteins bearing either the native human t-PA signal or the yeast acid phosphatase signal. Like the E. coli transformants of Harris et al., yeast were found to shunt most of the expressed protein into an insoluble, enzymatically-inactive reservoir (according to Lemontt et al. this occurred as a result of an associative interaction of the t-PA with cell membranes) without the secretion of soluble t-PA into the culture medium. Lemontt et al. did not describe whether the N-termini of the t-PA prepro constructions were properly processed, nor whether the minor proportion of soluble t-PA that was obtained was originally lodged in the periplasm or the cytoplasm.
EP 123,544 reported the secretion of human t-PA by the use of the alpha factor signal. The t-PA was divided equally between the cells (20 .mu.g/l) and the cultured medium (20 .mu.g/l).
On the other hand, EP 177,343 observes that little or no human t-PA activity could be obtained from E. coli transformants in which DNA encoding the alkaline phosphatase signal was fused to DNA encoding mature t-PA under the transcriptional control of the alkaline phosphatase promoter.
The mechanism responsible for the apparent inability of bacteria to express and secrete human plasminogen activators in commercially significant quantities remain inapparent, although an apparent requirement for glycosylation of the human enzymes has been implicated. Belin et al. ("Eur. J. Biochem." 148:225-232 [1985]) reported the secretion of enzymatically active murine urokinase from E. coli host cells which apparently were transformed with murine preprourokinase, but native murine urokinase is not normally glycosylated. On the other hand, Opdenakker et al. ("Proc. Soc. Exp. Biol. Med." 182:248-257 [1986]) reported significant reductions in the enzymatic activity of secreted t-PA when oocyte transformants were incubated with tunicamycin in order to inhibit glycosylation, the residual enzymatic activity in the tunicamycin-treated oocyte preparations being attributable to partially glycosylated t-PA. Other studies reported in the same work demonstrated reductions in t-PA activity by 15 to 85% when glycosylated t-PA was digested with glycolytic enzymes. Consistent with these findings was an earlier report by Opdenakker et al. ("Eur. J. Biochem." 131:481-487 [1983]) that reticulocyte translation products of the t-PA gene were devoid of enzymatic activity, a phenomenon described by these authors as possibly the result of the inability of reticulocyte preparations to perform post-translational functions (processing and glycosylation). On the other hand, a report by Kagitani et al. ("FEBS" 189(1):145 [1985]) observes enzymatic activity in homogenates of E. coli transformants with DNA encoding des (1-44) "finger-domain" deleted variant t-PA.
If, as suggested by the art, secretion of enzymatically active plasminogen activators that normally are glycosylated will only occur when preplasminogen activators are processed by eukaryotic secretory mechanisms, then it should not be possible to recover commercially significant amounts of secreted enzymatically active plasminogen activators from prokaryotic cell structure.
Accordingly, it is an objective herein to identify problems responsible for the failure of certain host vector systems to produce desired polypeptides, in particular to stably secrete mature polypeptides from certain preproteins. Realization of this objective will lead to host-vector systems which will successfully and routinely secrete mature heterologous polypeptides.
An additional objective is to devise a method for the secretion of fully functional human plasminogen activators from prokaryotic cell culture.
It is further objective to provide a method for the secretion of human tissue plasminogen activator (ht-PA) from bacterial cell culture in commercially significant quantities.
In the method of this invention inducible transcriptional control system is used to express a desired host cell-incompatible heterologous polypeptide in recombinant host cell culture wherein the degree of constitutive expression of the polypeptide is balanced against cell incompatibility.
These and other objects of the invention will be apparent from the specification as a whole.